Platelets are short-lived anucleate cells essential for primary hemostasis and recognized for their functions in thrombosis, immunity, antimicrobial defense, neurodegeneration, as well as cancer growth and metastasis. Their brief lifespan in circulation is controlled by the removal of sialic acid residues from the platelet surface (desialylation) and also the mitochondrial apoptosis pathway, with high expression of the anti-apoptotic protein BCL-XL being required for platelet survival. This dependence on BCL-XL has prevented the clinical deployment of recently developed small molecule inhibitors of BCL-XL, which have promising activity in solid as well as liquid cancers but cause on-target thrombocytopenia. Here, we investigate the functional relationship between platelet desialylation and apoptosis to determine how cross-talk between these mechanisms may impact platelet lifespan. We find that platelets progressively lose sialic acid residues and become more primed for apoptosis while in circulation, resulting in aged platelets that are desialylated and highly prone to undergoing apoptosis. In addition, platelet desialylation via endogenous or exogenous factors directly increases their BCL-XL dependence and accelerates apoptosis, which can be reversed by treatment with the sialidase inhibitor DANA (2,3-dehydro-2-deoxy-N-acetylneuraminic acid). Notably, young platelets recently released into circulation are less primed for apoptosis and less dependent on BCL-XL for survival. Consistent with these changes in priming, platelets aged in vitro exhibit increasing expression of multiple pro-apoptotic proteins including BIM, BAK and PUMA along with increasing cleaved caspase 3. Leveraging the lower BCL-XL dependence of young platelets, stimulation of de novo platelet production with the thrombopoietin receptor agonist romiplostim prevents BH3 mimetic-induced thrombocytopenia in vivo and may prevent severe platelet loss in patients treated with BCL-XL inhibitors.

Introduction  Social determinants of health (SDOH) are associated with differential outcomes after pituitary tumor treatment. However, the specific impact of SDOH is not well characterized. One reason may be the lack of collection and reporting of sociodemographic variables in the literature. This study aims to evaluate the frequency of reporting and distribution of participants' sex, race, ethnicity, income, and education level within pituitary surgery literature. We will compare the reported clinical research population demographics to the 2020 U.S. census. Methods  A systematic review was performed by searching PubMed, Cochrane, and Embase databases for pituitary surgery clinical research published between July 1, 2021 to June 30, 2022. We excluded studies that lacked a comparison group, were not original research (i.e., systematic reviews, meta-analysis), or included national databases and registry data. Results  The final analysis included 92 studies. A total of 99% of studies collected data on subject sex. On average 49% (range: 14-100%) of study populations were male. Only 4% ( n  = 4) studies included racial demographic data. Two studies included information on participants' ethnicity and two included education background. No studies included income or insurance data. Four U.S. studies included demographic distribution, and the reported race and ethnicity percentages are similar to the U.S. 2020 census distribution. Conclusion  Most clinical pituitary research collects and reports data on participant sex. However, very few studies collect and report data on other sociodemographic variables that can play a role in outcomes. The lack of sociodemographic information in clinical research literature makes it difficult to determine the role of SDOH on pituitary surgery outcomes.

Changes in surface glycan determinants, specifically sialic acid loss, determine platelet life span. The gradual loss of stored platelet quality is a complex process that fundamentally involves carbohydrate structures. Here, we applied lipophilic extraction and glycan release protocols to sequentially profile N-and O-linked glycans in freshly isolated and 7-day room temperature-stored platelet concentrates. Analytical methods including matrix assisted laser desorption/ionization time-of-flight mass spectrometry, tandem mass spectrometry, and liquid chromatography were used to obtain structural details of selected glycans and terminal epitopes. The fresh platelet repertoire of surface structures revealed diverse N-glycans, including high mannose structures, complex glycans with polylactosamine repeats, and glycans presenting blood group epitopes. The O-glycan repertoire largely comprised sialylated and fucosylated core-1 and core-2 structures. For both N-and O-linked glycans, we observed a loss in sialylated epitopes with a reciprocal increase in neutral structures as well as increased neuraminidase activity after platelet storage at room temperature. The data indicate that loss of sialylated glycans is associated with diminished platelet quality and untimely removal of platelets after storage.

Abstract Coactivator-associated arginine methyltransferase 1 (CARM1) is overexpressed in cancer, and it has emerged as an important target in acute myeloid leukemia and other hematologic malignancies. Janus kinase 2 (JAK2), that is activated by mutation in a variety of myeloid malignancies, can dictate chromatin structure via multiple effects. Here, we find that the hyperactivated JAK2-V617F mutant kinase phosphorylates CARM1, increasing its methyltransferase activity and altering its target specificity. Phospho-CARM1 binds and methylates the RUNX1 transcription factor, and the asymmetric dimethylation of R223 and R319 in RUNX1 is lost in engineered to express only non-phosphorylatable CARM1 mutant proteins in JAK2-V617F+ cell lines. The decreased CARM1 activity found in these cell lines impairs cell-cycle progression and induces apoptosis. We have established a link between activated JAK2 and CARM1 activity, and demonstrate that dual targeting of JAK2 and CARM1 is more effective than monotherapy in phospho-CARM1+ cell lines.

The session on the hemostatic system focused on new developments in coagulation and platelet biology as well as how therapeutic agents may affect hemostasis. The classic cascade model of coagulation was compared with the more recent models of cell-based and vascular-based coagulation, which may provide better insight on how the coagulation cascade works in vivo. A review of platelet biology highlighted that, as platelets age, desialylated platelets form and are recognized by Ashwell-Morell receptor (AMR), leading to hepatic uptake and subsequent increase in thrombopoietin (TPO) production. Administration of therapeutics that induce thrombocytopenia was also discussed, including Mylotarg, which is an antibody-drug conjugate that was shown to decrease human megakaryocyte development but had no effect on platelet aggregation. An acetyl co-A carboxylase inhibitor was shown to cause thrombocytopenia by inhibiting de novo lipogenesis, which is critical for the formation of the megakaryocyte demarcation membrane system responsible for platelet production. It was also illustrated how preclinical translation models have been very helpful in the development of adeno-associated virus (AAV) hemophilia B gene therapy and what old and new preclinical tools we have that can predict the risk of a prothrombotic state in people.

Introduction Coactivator-associated arginine methyltransferase 1 (CARM1) catalyzes the asymmetric dimethylation of arginine residues in histone and non-histone substrates, including transcription factors, RNA splicing factors, and the SWI/SNF chromatin remodeling complex. CARM1 has emerged as an important target in acute myeloid leukemia. We have previously shown that Janus kinase 2 (JAK2) affects the activity of epigenetic modifiers. A V617F mutation in JAK2 genes leads to its hyperactivation. This mutation is a common hallmark of the BCR/ABL1-negative myeloproliferative neoplasms, and also found in patients with acute myeloid leukemia and myelodysplastic syndrome. Here, we find that hyperactivated JAK2, mediated by biallelic V617F mutation, triggers CARM1 phosphorylation, increasing its methyltransferase activity and altering its target specificity. Methods We generated two phospho-tyrosine specific antibodies against CARM1 tyrosine-149 (Y139) and -334 (Y334), and two asymmetric dimethylation-arginine specific antibodies against RUNX1 arginine-223 (R223) and -319 (R319). We also generated isogenic HEL cells carrying homozygous CARM1 non-phosphorylatable mutation (Y149F or Y334F single, or Y149F/Y334F double mutation), using the CRISPR/Cas9 nuclease system. Results We first performed cell-free in vitro kinase assays using recombinant CARM1 protein and JAK2 kinase, and mass spectrometry analysis, which identified Y139 and Y334 as the sites of JAK2 phosphorylation in CARM1. We next compared the levels of CARM1-Y149 and -Y334 phosphorylation in 15 myeloid leukemia cell lines and normal human CD34+ cord blood cells. HEL and UKE-1 cells, that harbor bi-allelic JAK2-V617F mutation, showed the highest level of phosphorylated CARM1-Y149 and -Y334, while SET2 cells with mono-allelic JAK2-V617F mutation and the other cell lines showed abundant CARM1 protein but a lower relative amount of phosphorylated CARM1 protein. We found that the JAK2 activation, through transphosphorylation by JAK2 and TYK2, enhances tyrosine kinase activity against CARM1 using HEL cells with knockout of either JAK1 or TYK2. Having previously shown that CARM1 methylates RUNX1 at R223, in an immunoprecipitation assay, WT-CARM1 was able to pull down RUNX1, while the non-phosphorylatable CARM1 mutants were not. Using mass spectrometry and an in vitro methylation assay, we confirmed R223 and R319 as CARM1 target sites. We examined non-phosphorylatable CARM1 mutation knock-in HEL cells and found that mutation of either site reduces the asymmetric dimethylation of RUNX1 as well as known substrates (BAF155 and PABP1). We observed that CARM1 Y149F/Y334F mutant-expressing HEL cells reduced cell proliferation with G2/M cell cycle arrest and an increase in apoptosis. To identify the transcription networks regulated by CARM1 phosphorylation, we performed RNA-seq on WT-CARM1 and double CARM1 Y149F/Y334F mutant-expressing HEL cells. Gene set enrichment analysis identified that gene sets associated with G2/M cell cycle progression and anti-apoptosis were significantly downregulated in Y149F/Y334F mutation knock-in HEL cells. We next performed ChIP-seq analyses using antibodies against total RUNX1 and against asymmetrically dimethylated R319-RUNX1 in HEL cells expressing knock-in of CARM1 non-phosphorylatable mutation. CARM1 non-phosphorylatable mutation decreased the signals of dimethylated RUNX1-R319 within 5 kb of the transcription start sites for RUNX1 target genes, with less effect on total RUNX1. We next assessed the efficacy of the combination with CARM1 inhibitor (EPZ025654) and JAK2 inhibitor (ruxolitinib). Dual targeting inhibition of JAK2 and CARM1 showed a synergistic inhibition effect on HEL and UKE-1 cells (with abundant phosphor-CARM1), but only an additive effect on SET2 cells (with less amount of phosphor-CARM1). Conclusion CARM1 phosphorylation mediated by hyperactivated JAK2 regulates its methyltransferase activity and is required for the maximal proliferation of myeloid neoplasms. We also demonstrated that dual targeting of JAK2 and CARM1 is more effective than monotherapy in cell lines with phosphor-CARM1. Thus, our results reveal a novel role for CARM1 phosphorylation in myeloid neoplasms.

Immune thrombocytopenia (ITP) is a platelet disorder. Pediatric and adult ITP have been associated with sialic acid alterations, but the pathophysiology of ITP remains elusive, and ITP is often a diagnosis of exclusion. Our analysis of pediatric ITP plasma samples showed increased anti-Thomsen-Friedenreich antigen (TF antigen) antibody representation, suggesting increased exposure of the typically sialylated and cryptic TF antigen in these patients. The O-glycan sialyltransferase St3gal1 adds sialic acid specifically on the TF antigen. To understand if TF antigen exposure associates with thrombocytopenia, we generated a mouse model with targeted deletion of St3gal1 in megakaryocytes (MK) (St3gal1MK-/-). TF antigen exposure was restricted to MKs and resulted in thrombocytopenia. Deletion of Jak3 in St3gal1MK-/- mice normalized platelet counts implicating involvement of immune cells. Interferon-producing Siglec H-positive bone marrow (BM) immune cells engaged with O-glycan sialic acid moieties to regulate type I interferon secretion and platelet release (thrombopoiesis), as evidenced by partially normalized platelet count following inhibition of interferon and Siglec H receptors. Single-cell RNA-sequencing determined that TF antigen exposure by MKs primed St3gal1MK-/- BM immune cells to release type I interferon. Single-cell RNA-sequencing further revealed a new population of immune cells with a plasmacytoid dendritic cell-like signature and concomitant upregulation of the immunoglobulin rearrangement gene transcripts Igkc and Ighm, suggesting additional immune regulatory mechanisms. Thus, aberrant TF antigen moieties, often found in pathological conditions, regulate immune cells and thrombopoiesis in the BM, leading to reduced platelet count.

The Thomsen-Friedenreich antigen (TF-antigen) occurs during exposure of the underlying Core-1 disaccharide (Gal-beta(1,3)GalNAc) through the loss of its capping sialic acid (Sia). Exposure of the cryptic TF-antigen occurs during inflammation, during acute infections with influenza viruses or bacteria, in malignancies, and is associated with thrombocytopenia. Exposure of the TF-antigen on circulating blood cells, including platelets and red blood cells (RBC), can lead to severe thrombocytopenia or hemolysis in hemolytic uremic syndrome and other immune diseases. Recent data suggest that altered Sia may cause platelet destruction because treatment with the sialidase inhibitor Tamiflu increases platelet count in healthy and thrombocytopenic patients. In humans, genetic mutations involving Sia synthesis and transport, and atypical cell surface sialylation, unrelated to any genetic mutation, are associated with reduced platelet count, supporting the role of Sia in regulating platelet count. Immune cells, including classical dendritic cells (cDCs), plasmacytoid dendritic cells (pDCs), and subsets of T cells (CD8+, CD4+, and Treg cells) can also affect immune thrombocytopenia pathogenesis. Like many other immune cells, cDCs, and pDCs express Siglecs (sialic-acid-binding immunoglobulin-like lectins), which often contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that act as immunosuppressors. Whether BM immune cells monitor MKs via glycan-lectin receptors, including Siglecs and Sia interactions, to control platelet production is unclear. To investigate the role of the TF-antigen in thrombopoiesis, we generated St3gal1MK-KO mice (Pf4-Cre) that display increased TF-antigen specifically in megakaryocytes (MK) and platelets. St3gal1MK-KO mice developed significant thrombocytopenia, but had normal platelet half-life, suggesting that the TF-antigen affected BM thrombopoiesis. In vitro MK maturation and proplatelet production from primary ST3Gal1MK-KO mouse BM cells were also normal, pointing to extrinsic factors in the BM environment affecting thrombopoiesis. Platelet counts of St3gal1MK-KO mice were restored to wild-type levels by 1) crossing St3gal1MK-KO mice with Jak3KO mice that have impaired of lymphoid cell development, 2) by treatment with anti-inflammatory dexamethasone, and 3) treatment with a depleting anti-CD4 antibody. Immunofluorescence staining of the St3gal1MK-KO BM revealed proplatelet structures positive for GPIba+ and the TF-antigen, being infiltrated by mononuclear cells resembling lymphocytes. We speculated that immune cells surveil megakaryocytes to control thrombopoiesis. Bulk RNAseq of CD4+ cells in St3gal1MK-KO BM confirmed a population bias for Type I interferon (IFN-I)-releasing pDCs, a cell type regulated by unique sialic acid binding lectins (Siglecs). Inhibition of IFN-I activity, by a blocking receptor antibody improved platelet counts in St3gal1MK-KO mice. Co-cultures of pDCs with MKs show inhibited pro-platelet formation when TF-antigen is present on MKs with elevated IFN-I levels. Gene set enrichment analysis of BM pDCs single cell RNASeq (scRNAseq) data further confirmed that TF-antigen exposure by MKs up-regulates IFN-I transcripts. scRNAseq also reveals a new population of immune cells with pDC transcript signature and concomitant upregulation of immunoglobulin re-arrangement gene transcripts Igkc and Ighm. In conclusion, the data shows that recognition of aberrant MK sialylation by pDCs regulates thrombopoiesis through IFN-I secretion. Disclosures No relevant conflicts of interest to declare.

Our understanding of cell biological processes involved in aging has advance greatly over the past decades. Platelets are small cells that circulate for 4-5 days in mice and 7-10 days in humans. And even though, platelets are anucleated cells, a growing body of evidence shows that platelet clearance is a well-regulated mechanism. We have recently demonstrated that platelets lose sialic acid as they circulate and age in blood and are rapidly cleared by the hepatic Ashwell-Morell receptor (AMR). And others have shown, in a series of studies using genetically modified mice or pharmacological inhibitors that platelets undergo apoptosis by triggering the intrinsic mitochondrial apoptotic machinery. Here, we investigate if desialylation and apoptosis are related events. First, using a newly developed state-of-the-art technique called dynamic BH3 profiling (DBP), we investigated the mitochondria readiness to undergo apoptosis on platelets derived from WT and AMR deficient (Asgr2-/-) mice. In our assay, digitonin-permeabilized platelets were exposed to activators signaling peptides (such as Bim, Bid and PUMA), and as cells undergo apoptosis due to peptide treatment, they released Cytochrome C. Our data showed that desialylated platelets derived from Asgr2-/-mice have high background levels of Cytochrome C release when compared to WT platelets in the presence of all activator peptides, indicating that desialylated platelets are highly primed to apoptosis. We also tested the level of dependence on pro-survival protein, by using sensitizer peptides (Bad, Hrk and MS1). We observed that desialylated platelets (Asgr2-/-platelets), and to a certain degree, WT platelets, are extremely sensitive to BCL-xL inhibition, as indicated by the extremely high response to Bad and Hrk peptides even at lower concentrations (0.1 and 1uM). Surprisingly, WT and Asgr2-/-platelets show very little response to the MS1 peptide, indicating that they are not dependent on MCL1 for survival, as otherwise suggested. Flow cytometry analysis revealed desialylated platelets from Asgr2-/-mice have a ~2-fold increase in Phosphatidylserine (PS) surface exposure when compared to WT platelets. In addition, western blot analysis showed increased expression of cleaved caspase 3 in Asgr2-/-platelets, but no changes in Bcl-xL protein expression between WT and Asgr2-/-platelets. Next, WT and Asgr2-/-mice received a single dose of the BH3 mimetic, ABT-737, which binds and inhibits pro-survivor proteins (Bcl-2, Bcl-xL and Bcl-w) inducing apoptosis in vivo. Approximately 2 hours after the injection of ABT-737, we observed a big drop on platelets counts in both WT (~42%) and Asgr2-/-(~59%) mice. Importantly, platelets from Asgr2-/-mouse were cleared more efficiently (~20%) from the circulation when compared to those in WT mice, consistent with the ~20% increment in platelet number observed in this mouse model and supporting the notion that the platelets that circulate longer in the Asgr2-/-mice are more sensitive to apoptotic events. To investigate if apoptosis could be triggering platelet desialylation, WT mice were treated with ABT-737 and 1hour later (time point before platelet count drop), platelets were collected and analyzed by flow cytometry. Interestingly, analysis of galactose exposure by RCA-I lectin showed no differences in desialylation between ABT-737 and PBS control groups. On the other hand, Phosphatidylserine (PS) exposure was significantly elevated on ABT-737 group, indicating that platelets were undergoing apoptosis without changing their sialylated status. To confirm our in vivodata, freshly isolated washed WT platelets were treated with ABT-737 to induce apoptosis or Neuraminidase (NA) to desialylated platelets. NA treatment induced platelet desialylation (increased RCA-I binding) in WT platelets, as expected, and interestingly triggered apoptosis, judge by increased PS exposure in both ABT-737 and NA treated groups. However, ABT-737 treatment wasn't able to induce desialylation as levels of RCA-I binding to platelets was the same when compared to PBS control platelets. Taken together, our data shows that desialylated platelets in circulation are prone to apoptosis. In addition, our findings strongly support the hypothesis that desialylation of platelet surface glycoproteins trigger the intrinsic apoptotic pathway in platelets in vivo. Disclosures No relevant conflicts of interest to declare.

Platelets are fundamentally important in normal hemostasis and pathological thrombosis (i.e. cardiovascular diseases, stroke, etc.). Platelets mediate the initial first-step in hemostasis through surface glycoproteins like the GPIb-IX-V complex and integrin αIIbβ3 (GPIIbIIIa). Although the functions of platelet surface glycoproteins are well known, the roles of posttranslational modifications on those surface glycoproteins are poorly understood. We have recently shown that sialic acid is a key regulator of platelet survival. As platelets circulate and age in blood, they lose sialic acid and are rapidly cleared by the hepatocytes where they stimulate liver TPO production and consequently regulate thrombopoiesis. Here, we investigated the importance of glycosylation to platelet function by measuring the impact of sialic acid content on platelet responses to thrombin activation.

Patients with Essential Thrombocythemia (ET) have abnormal platelet counts and function leading to increased thrombo-hemorrhagic events, which are the principal causes of morbidity and mortality in these patients. ET results from somatic mutations altering genes involved in intracellular signaling pathways: mutations in the JAK2 kinase gene are found in 50-60% of patients, mutations in the calreticulin (CALR) represents 30-40% of patients and mutations in MPL, the thrombooietin receptor, represents 5-10% of patients. JAK2 mutations lead to a constitutive activation of intracellular signaling pathway of JAK-STAT resulting in hyperactivated platelets. CALR has been shown to associate with MPL, the thrombopoietin receptor, and consequently activates its downstream signaling via JAK-STAT signaling. Interestingly, CALR -mutant patients have a better prognosis than JAK2 -mutant patients with less thrombotic events regardless of the platelet count. Despite the high frequency of CALR and JAK2 mutations, the mechanisms by which these mutations promote thrombosis are unknown. In this study, we characterized the platelet glycoprotein profile and investigated the molecular mechanisms leading to platelet activation of ET patients according to JAK2 or CALR mutations.

Glycosyltransferases, usually residing within the intracellular secretory apparatus, also circulate in the blood. Many of these blood-borne glycosyltransferases are associated with pathological states, including malignancies and inflammatory conditions. Despite the potential for dynamic modifications of glycans on distal cell surfaces and in the extracellular milieu, the glycan-modifying activities present in systemic circulation have not been systematically examined. Here, we describe an evaluation of blood-borne sialyl-, galactosyl- and fucosyltransferase activities that act upon the four common terminal glycan precursor motifs, GlcNAc monomer, Gal(β3)GlcNAc, Gal(β4)GlcNAc and Gal(β3)GalNAc, to produce more complex glycan structures. Data from radioisotope assays and detailed product analysis by sequential tandem mass spectrometry show that blood has the capacity to generate many of the well-recognized and important glycan motifs, including the Lewis, sialyl-Lewis, H- and Sialyl-T antigens. While many of these glycosyltransferases are freely circulating in the plasma, human and mouse platelets are important carriers for others, including ST3Gal-1 and β4GalT. Platelets compartmentalize glycosyltransferases and release them upon activation. Human platelets are also carriers for large amounts of ST6Gal-1 and the α3-sialyl to Gal(β4)GlcNAc sialyltransferases, both of which are conspicuously absent in mouse platelets. This study highlights the capability of circulatory glycosyltransferases, which are dynamically controlled by platelet activation, to remodel cell surface glycans and alter cell behavior.

The human body produces and removes 1011 platelets daily to maintain a normal steady-state platelet count. However, the regulatory mechanisms remain elusive. We have shown that platelets lacking sialic acid (desialylated platelets) are removed by the hepatic Ashwell-Morell receptor (AMR or asialoglycoprotein receptor type 2), thereby regulating platelet survival and hepatic TPO levels. Platelet counts and lifetime were increased in Asgr2-/- mice (AMR-null mice), compared to wild type (WT) mice. Platelet volume and immature platelet fraction (IPF) are decreased in AMR-null mice, consistent with the notion that platelets in AMR-null mice (AMR-null platelets) circulate longer and are older.

Bone marrow (BM) macrophages maintain both survival and retention of hematopoietic stem cells and regulate erythropoiesis. The role of macrophage lectins and glycans in thrombopoiesis remains unclear. We report a novel role for bone marrow macrophage galectin-3 in maintaining platelet counts, by phagocytosing megakaryocytes (MKs) expressing the Thomsen-Friedenreich (TF) antigen, which is often exposed under pathological conditions, such as cancer and malignancies.

The human body produces and removes 1011 platelets daily to maintain a normal steady state platelet count. Platelet production must be regulated to avoid spontaneous bleeding or arterial occlusion and organ damage. Multifaceted and complex mechanisms control platelet production and removal in physiological and pathological conditions. This review will focus on different mechanisms of platelet senescence and clearance with specific emphasis on the role of posttranslational modifications. It will also briefly address platelet transfusion and the role of glycans in the clearance of stored platelets.

The human body produces and removes 10 11 platelets daily to maintain a normal steady‐state platelet count. Platelet production must be tightly regulated to avoid spontaneous bleeding if counts are low (thrombocytopenia) or arterial occlusion and organ damage if counts are high (thrombocytosis). Recent studies have highlighted the role of glycan modifications on platelet surface proteins in mediating platelet clearance. Platelets undergoing cold storage lose sialic acid and β ‐galactose. Following transfusion, cold‐stored platelets are cleared via hepatic A shwell– M orell receptor ( AMR ) and the macrophage ( K upffer cell) α M β 2 integrin lectin domain. New data point to the fact that platelets surface loses sialic acid during their circulatory lifetime, suggesting a novel platelet in vivo clearance mechanism via the AMR . Thrombopoietin ( TPO ) is the primary regulator of platelet production, supporting the survival, proliferation and differentiation of the platelet precursors, bone marrow megakaryocytes. Although it is clear that hepatocytes are the primary source of TPO , the mechanisms regulating circulating TPO levels have been subject to discussion for decades. Two major models for TPO regulation have been proposed. In one model, hepatic TPO production is constitutive and TPO serum levels are maintained solely by its uptake and metabolism by the TPO receptor c‐ M pl on platelets and megakaryocytes. In another model, hepatic TPO production is regulated. However, the physiological ligand–receptor pair capable of regulating steady‐state TPO production remains unclear. In this review, mechanisms of glycan–lectin‐mediated platelet clearance and TPO production will be discussed.

Immune thrombocytopenia (ITP) is a common bleeding disorder caused primarily by autoantibodies against platelet GPIIbIIIa and/or the GPIb complex. Current theory suggests that antibody-mediated platelet destruction occurs in the spleen, via macrophages through Fc-FcγR interactions. However, we and others have demonstrated that anti-GPIbα (but not GPIIbIIIa)-mediated ITP is often refractory to therapies targeting FcγR pathways. Here, we generate mouse anti-mouse monoclonal antibodies (mAbs) that recognize GPIbα and GPIIbIIIa of different species. Utilizing these unique mAbs and human ITP plasma, we find that anti-GPIbα, but not anti-GPIIbIIIa antibodies, induces Fc-independent platelet activation, sialidase neuraminidase-1 translocation and desialylation. This leads to platelet clearance in the liver via hepatocyte Ashwell-Morell receptors, which is fundamentally different from the classical Fc-FcγR-dependent macrophage phagocytosis. Importantly, sialidase inhibitors ameliorate anti-GPIbα-mediated thrombocytopenia in mice. These findings shed light on Fc-independent cytopenias, designating desialylation as a potential diagnostic biomarker and therapeutic target in the treatment of refractory ITP.

Dynamins are highly conserved large GTPases (enzymes that hydrolyze guanosine triphosphate) involved in endocytosis and vesicle transport, and mutations in the ubiquitous and housekeeping dynamin 2 (DNM2) have been associated with thrombocytopenia in humans. To determine the role of DNM2 in thrombopoiesis, we generated Dnm2(fl/fl) Pf4-Cre mice specifically lacking DNM2 in the megakaryocyte (MK) lineage. Dnm2(fl/fl) Pf4-Cre mice had severe macrothrombocytopenia with moderately accelerated platelet clearance. Dnm2-null bone marrow MKs had altered demarcation membrane system formation in vivo due to defective endocytic pathway, and fetal liver-derived Dnm2-null MKs formed proplatelets poorly in vitro, showing that DNM2-dependent endocytosis plays a major role in MK membrane formation and thrombopoiesis. Endocytosis of the thrombopoietin receptor Mpl was impaired in Dnm2-null platelets, causing constitutive phosphorylation of the tyrosine kinase JAK2 and elevated circulating thrombopoietin levels. MK-specific DNM2 deletion severely disrupted bone marrow homeostasis, as reflected by marked expansion of hematopoietic stem and progenitor cells, MK hyperplasia, myelofibrosis, and consequent extramedullary hematopoiesis and splenomegaly. Taken together, our data demonstrate that unrestrained MK growth and proliferation results in rapid myelofibrosis and establishes a previously unrecognized role for DNM2-dependent endocytosis in megakaryopoiesis, thrombopoiesis, and bone marrow homeostasis.

Glycosylation defects have been associated with low platelet counts. Six genes encoding sialyltransferases (ST), ST3gal1 to 6, that synthesize an α2,3 sialic acid (SA) linkage have been identified in the mammalian genome, and deletion of St3gal1 and St3gal4 genes has been associated with macrothrombocytopenia in mice. Despite the similarity in transferring SA in a α2,3-linkage to terminal galactose residues, St3gal1 and St3gal4 sialylate distinct glycans: St3gal1 is associated with core 1 O-glycan Galβ1,3GalNAcα1-Ser/Thr expression, also known as tumor-associated or Thomsen-Friedenreich antigen (T-antigen), whereas St3gal4 sialylates lactosaminyl Galβ1,4GlcNAc N-glycans. It has been previously shown that St3gal4-null platelets are cleared by the hepatic Ashwell-Morell receptor, causing severe thrombocytopenia in these mice. Herein, we generated St3gal1loxP/PF4+ mice specifically lacking ST3Gal1 in the megakaryocyte (MK) lineage to investigate the detailed mechanisms of macrothrombocytopenia associated with St3gal1 deficiency. Both St3gal1loxP/PF4+ circulating platelets and bone marrow (BM) MKs had increased T-antigen expression, compared to control, as evidenced by peanut agglutinin (PNA) binding. As expected, other blood cell lineages had no increase in T-antigen expression. Blood platelet counts were reduced by ~50% and platelets were enlarged in St3gal1loxP/PF4+ mice, compared to control, despite a virtually indistinguishable platelet clearance. BM MK numbers were normal despite the observed thrombocytopenia, BM MK colony forming units (CFUs) were reduced and in vitro proplatelet production was normal in St3gal1loxP/PF4+ mice, suggesting that extrinsic factors in the St3gal1loxP/PF4+ BM environment affected platelet production. We hypothesize that recognition of the T-antigen epitope on MKs mediate phagocytosis by macrophages. Macrophages in St3gal1loxP/PF4+ mice had increased expression of CD68 (macrosialin), indicative of an activated macrophage state. Flow cytometric analysis of BM derived macrophages of St3gal1loxP/PF4+ mice showed an increased population of resolving M2-type macrophages, which are normally involved in apoptotic cell clearance. Additionally, St3gal1loxP/PF4+ BM smears revealed increased hemophagocytosis, as evidenced by May-Grunwald/Giemsa, indicative of an unspecific increase in phagocytic macrophages. Macrophage ablation by in vivo injection of clodronate-encapsulated liposomes significantly reduced the numbers of activated macrophages in St3gal1loxP/PF4+ mice, thereby normalizing blood platelet counts and size. Taken together data show the contrasting effects of different SA loss on platelet homeostasis: Platelets lacking α2,3-linked SA on N-glycans have increased platelet clearance, whereas a lack of α2,3-linked on O-glycans do not affect platelet half-life, but cause defective thrombopoiesis in MKs. Disclosures No relevant conflicts of interest to declare.